Retinal Thickness and P-ERG Changes in Adult Patients With Anisometropic and Strabismic Amblyopia

Objective To investigate the changes of retinal thickness and P-ERG signals in adult patients with anisometropic and strabismic amblyopia. Methods Sixty patients with monocular adult amblyopia, including 30 anisometropic amblyopes (AA group) and 30 strabismic amblyopes (SA group), were enrolled in our study at the outpatient clinic of The Hefei First People’s Hospital Hospital of Anhui medical University from June 2019 to November 2020. Retinal nerve ber layer (RNFL) thickness was measured within 3.4 mm diameter range surrounding the optic nerve, and ganglion cell complex (GCC) layer thickness within 6 mm diameter range surrounding the fovea by an Optovue RTVue OCT in both amblyopic and fellow eyes. The amplitude and latency of P 50 and N 95 in P-ERG were recorded by a Roland electrophysiology instrument under two stimulation conditions with different temporal and spatial frequencies that were designed to bias the parvocellular and magnocellular pathways respectively. Data between amblyopic and fellow eyes was statistically analyzed by paired t test. The correlation between axial length and parameters of OCT and P-ERG was examined by Pearson correlation test. Our results showed that the alterations in structure and function of retina that could be seen in adult anisometropic amblyopia were not found in adult strabismic amblyopia group. The functional loss in anisometropic amblyopia was found to bias to a damage of partial ganglion cells by the parvocellular pathway. These ndings indicated that the pathological mechanisms were different between anisometropic and strabismic amblyopia.


Introduction
Abnormal visual input in critical period, commonly due to anisometropia or strabismus, results in amblyopia (mostly unilateral), whose visual acuity can't be corrected to normal, with an incidence rate of 1-5% [1]. Causes for amblyopia include strabismus, anisometropia, form deprivation, and uncorrected refractive errors during the sensitive period of visual system development, but the pathophysiological mechanism of amblyopia is still unclear [2]. Von Noordent et al. [3] and Wiesel TN et al. [4], through animal experiments, found that amblyopia could affect the development of lateral geniculate body cells, leading to abnormal cell morphology. These geniculate body neurons are responsible for receiving the eyes' visual signals and project signals onto the visual cortex. Previous pattern reversal visual evoked potentials (PVEP) studies also showed that the amplitude and latency time of P 100 wave were signi cantly decreased in amblyopic eyes [5,6]. These studies revealed that in amblyopia, structural and functional alterations could be seen in the visual cortex and subcortical area.
Are there any structural and functional changes that can be found in the retina in patients with amblyopia? In recent years, the wide application of the spectral domain OCT (SD-OCT) has made it possible to measure the thickness of the retinal nerve ber layer. Altintas et al. [7], Al-Haddad et al. [8], with the use of an OCT, found no signi cant difference in retinal nerve ber layer (RNFL) thickness in patients with anisometropic amblyopia when comparing their amblyopic eyes with the fellow eyes.
However, Yoon et al. [9] also used OCT to examine children ages 5 to 12 with anisometropic amblyopia and found that these patients' RNFL thickness increased in the amblyopic eyes when compared to the fellow healthy eyes. The above-mentioned research re ects contradictory results of RNFL thickness changes in amblyopic eyes. The present consensus regarding structural changes in the retina of patients with amblyopia mainly focuses on the ganglion cells alternations after birth [10,11]. The ganglion cell complex (GCC), comprising the nerve ber layer, ganglion cell layer, and inner plexiform layer, is a good indicator of ganglion cells status. Park et al. [12] and Tugcu et al. [13] measured amblyopic patients' GCC thickness but arrived at opposite results. Park et al. found GCC thickness in amblyopic eyes reduced when compared to fellow healthy eyes. Tugcu et al., on the other hand, found that patients with anisometropic amblyopia had a signi cant increase in GCC thickness in the amblyopic eyes.
Pattern-electroretinogram (P-ERG) re ects the function of retina, especially the ganglion cells function. Arden et al. [14] found that P 50 amplitude of amblyopia eyes in 6 to 28 year old patients with anisometropic and ametropic amblyopia is smaller than that of normal control eyes; but other researchers such as Guttob et al. [15] and Hess et al. [16] reported that the amblyopic eyes, comparing to normal control group, showed no signi cant difference in P-ERG in any type of amblyopia. Whether or not structural or functional changes of the retinal are present in amblyopia has been of interest to many researchers, but results have been con icting. Three reasons may be behind this, including: (1) Age factor: Some studies were based on children's visual sensitive period for the study.
Children's visual development and plasticity are different from those of an adult, and so direct comparison may produce con icting conclusions; (2) Different types of amblyopia become a confounding factor; and (3) Racial differences and potential measurement errors are also considered as potential confounding factors [17].
With the goal to explore retinal changes of adult amblyopia, we used OCT and PERG to investigate the functional and structural alternations of retinal in anisometropic and strabismic amblyopes.

Subjects And Methods
Participants 30 cases of adult anisometropic amblyopia (AA) and 30 cases of adult strabismic amblyopia (SA), age 21.35 ± 2.14 years (Mean ± SD), were recruited at the eye outpatient clinic of the Hefei First People's Hospital of Anhui medical University from June 2019 to November 2020. All subjects were of Chinese origin with a male to female ratio close to 1:1. The visual acuity of the amblyopic eyes in the AA group was 0.509 ± 0.333 logMAR, and in the SA group, 0.611 ± 0.277 logMAR. The study was approved by the ethics committee of the Hefei First People's Hospital of Anhui medical University University and followed the Declaration of Helsinki. Written informed consent was obtained from all subjects.
The subjects' best corrected visual acuity (BCVA) in the amblyopic eye was required to be ≥ 0.222 logMAR, and the fellow healthy eye's BCVA was required to be ≤ 0 logMAR. Subjects were excluded if there was any anterior segment, fundus disease, or previous ocular surgery, or if there was any systemic disease that may affect the eyes. Both amblyopic and fellow eye in each subject were required to have normal central xation.
In the AA group, only subjects with a refractive error difference of spherical power greater than 3.0D between the two eyes, and cylindrical power difference of greater than 1.0D, were included. In the SA group, subjects with an interocular difference of spherical power ≤ 1.5D, and cylindrical power difference of < 1.0D, were included. Characteristics of AA and SA patients are shown in Table 1. Data were presented as mean ± SD. Data were presented as Mean ± SD. The tabular dates are presented as Mean ± SD. No signi cant correlation was found between axial length and the results from the OCT or P-ERG (Pearson's Correlation, p > 0.05).
Examination methods

General Examination:
The anterior segment was examined by a slip-lamp, and the fundus was checked with a direct ophthalmoscopy. BCVA was measured with the Snellen chart at a distance of 5 m and converted into logMAR values for statistical analysis. Intraocular pressure was measured by applanation tonometry prior to instillation of tropicamide eye drops, three times in total. Axial length (AL) was measured with a Lenstar LS 900 (LS 900® Haag-Streit AG, Koeniz, Switzerland). Cover test with prism was applied to identify the presence of strabismus and the degree of strabismus.

OCT Examination:
Optovue RTVue (RTVue-100, version 2.0.4.0, Optovue Inc., Fremont, Canada) Fourier-domain OCT imaging technology was used in this study. OCT examination was completed with a natural pupil size, and by the same skilled examiner. RNFL thickness within a 3.4mm diameter range surrounding the optic nerve (ONH scan) and GCC thickness within 6mm diameter range using the fovea as the center (GCC scan) were measured in all amblyopic and fellow healthy eyes. The RNFL thickness of four sectors (T: temporal, S: superior, N: nasal, I: inferior) [18] was analyzed (Fig. 1a).

P-ERG Examination
The Roland electrophysiology instrument (ROLAND CONSULT, Color Ganzfeld 0450C, Germany) was utilized to record P-ERG. Subjects wore glasses to maintain BCVA and were asked to sit in a dim room (average luminance of 5 cd/m 2 ). Before the test, each subject was given 10 minutes for dark adaptation and a pupil diameter of about 5 mm was maintained with no mydriatic or miotic. Display contrast was 80%, and the average brightness was 110 cd/m 2 . Subjects were positioned 1 m away from the monitor.
The following two different P-ERG stimulation patterns were used: the magnocellular pathway biased stimulation modes: low spatial frequency (4c/d) -high temporal frequency (8 Hz) (LS-HT model); and the parvocellular pathway biased stimulation modes: high spatial frequency (12c/d) -low temporal frequency (1 Hz) (HS-LT model) [19]. Silver chloride skin electrodes were placed on both sides of the lower eyelid and temples as a recording line and forehead acted as a ground reference line. Each stimulus ipped 200 times in a row to be superimposed and recording electrical impedance remained below 5Ω.
The recording signal was ampli ed 50,000 times by the dual differential ampli ers. Filters were set between 1Hz and 30Hz. The computer automatically recorded the steady-state P-ERGs near sinusoidal by Fourier transformation. P 50 , N 95 amplitude and latency were analyzed.

Statistical Analysis
Statistical analysis was performed using SPSS 15.0 (SPSS Inc, Chicago, IL). Signi cant level of P values is ≤ 0.05. OCT and P-ERG parameters were compared by using paired t test. The correlation between the axial length (AL) and parameters of OCT, P-ERG was analyzed by using the Pearson correlation test.

Results
(1) Changes of RNFL thickness in anisometropic amblyopia and strabismic amblyopia patients: RNFL thickness of amblyopic eyes was signi cantly thinner in the AA group in the temporal sector than that of the fellow healthy eyes (n = 15, p < 0.05), but in the other three sectors (nasal, superior, and inferior), RNFL thickness signi cantly increased compared with the fellow eyes (n = 15, p < 0.05) by an OCT examination (Fig. 1b). In the SA group eyes, no statistically signi cant difference was found in RNFL thickness of the four sectors compared with the fellow healthy eyes (p > 0.05).
(2) Changes of GCC thickness in anisometropic amblyopia and strabismic amblyopia patients: GCC layer thickness in the amblyopic eyes signi cantly increased compared with the fellow healthy eyes in the AA group (n = 15, p < 0.05). But in the SA group, there was no statistically signi cant difference compared to the fellow eyes (p > 0.05) (Fig. 2).
(3) P-ERG ndings in anisometropic amblyopia and strabismic amblyopia patients. P-ERG in parvocellular pathway biased stimulated mode: Compared with the fellow healthy eyes, P 50 amplitude (n = 15, t = -3.329, p < 0.05) and N 95 amplitude (n = 15, t = -2.235, p < 0.05) signi cantly decreased, but there was no signi cant difference (p > 0.05) in latency in the AA group. While there was no signi cant difference in P 50 , N 95 amplitude and latency in the SA group (p > 0.05). P-ERG in magnocellular pathway biased stimulated mode: No signi cant difference was found in amplitude and latency in both the AA and SA groups (p > 0.05) ( Table 2). The table shows the latency and amplitude of P-ERG waves produced by the LS-HT and HS-LT stimulation in the AA group; the italic bold text shows the decrease of the amplitude of AA patients' P50 and N95 in the stimulations. The amplitude decrease of amblyopic eyes of AA patients is signi cantly different compared with the contralateral healthy eyes.
(4) There was no signi cant difference of AL between amblyopic eyes and fellow eyes both in the AA and SA groups (p > 0.05), and there was also no signi cant correlation between axial length and OCT, P-ERG parameters (Pearson correlation test).

Discussion
Amblyopia occurs when there are abnormal visual experiences during the sensitive period of visual development, leading to the reduction of one or both eyes' BCVA to below age standard. In this study, we used OCT and P-ERG to investigate retinal structural and functional changes in adult patients with monocular anisometropic or strabismic amblyopia. The analysis of RNFL thickness comprised of four regions and P-ERG was recorded and analyzed by two different modes, biasing the parvocellular pathway and magnocellular pathway respectively. We found GCC and all sectors of RNFL thickness except the temporal sector signi cantly increased and P 50 and N 95 amplitudes signi cantly reduced in the amblyopic eyes of the AA group compared with the fellow eyes while no signi cant changes of OCT and ERG parameters could be seen in SA group.
Inconsistent results have been reported by different scholars in observing the changes of retinal structure in amblyopia by using OCT. Altintas [7] et al. and Al-Haddad et al. [8] found that the RNFL thickness of amblyopic eyes was not signi cantly different from the contralateral healthy eyes in either adult or children patients with anisometropic amblyopia. Furhtermore, Firat et al. [20] reported that RNFL thickness in the amblyopic eyes of children was not signi cantly different from the healthy eyes in the normal control group. But Yoon et al. [9] found that RNFL was signi cantly thicker in the amblyopic eyes than the contralateral healthy eyes in anisometropic amblyopic children. We speculated that the cause of the inconsistency was that the total peripapillary RNFL had been analyzed in previous studies whereas different regions of RNFL might vary in amblyopia. So we analyzed RNFL in four sectors separately in this study and found RNFL thickness in the nasal, superior and inferior sectors signi cantly increased. At the same time, the temporal sector of RNFL, where the maculopapillary bundle is located, in amblyopic eyes is thinner. The maculopapillary bundle is believed to be responsible for the central visual signals from the macula to the optic nerve. Previous studies suggested that in amblyopia, central vision is impaired; in other words, damage occurs mainly in the center of the macula and central visual pathways with no signi cant change to peripheral vision when compared to the contralateral eyes. We speculated that the thinning of RNFL in the maculopapillary bundle may cause degeneration of central vision and corresponding excessive ganglion cell atrophy or apoptosis. Thickening in RNFL layer in other sectors might be due to the blocking of normal ganglion cells' apoptosis after birth [10].
Changes in ganglion cells after birth are considered to be one of main reasons for retinal structural alteration in amblyopia [10]. Apart from RNFL, the GCC layer includes inner plexiform layers, ganglion cell body, and nerve ber. Tugcu et al. [13], using OCT to measure GCC thickness, found that in strabismic amblyopia patients (aged 3-13 years), the GCC layer was thicker when compared to the fellow healthy eyes. However, Park et al. [12] found that patients with amblyopia had signi cantly reduced GCC layer average thickness than the contralateral healthy eyes. The present study found an increase in the thickness of the GCC layer in adult patients with anisometropic amblyopia compared with the contralateral eyes, but no statistically signi cant change in strabismic amblyopia. This is consistent with the study by Yen et al [10] that suggested that normal apoptosis of retinal ganglion cells was blocked after birth in amblyopia, which can lead to an increase in RNFL thickness. Szigeti A et al. [11] suggested that if Yen and others' hypothesis was correct, then the suppression of retinal ganglion cells' apoptosis would not only affect the thickness of the RNFL, but also result in a thickening of the GCC layer. These studies provided evidence that the mean GCC thickness would increase in amblyopia.
Besides the structural changes that could be observed in the amblyopic eyes, P-ERG was used in exploring the functional changes of the retina in our study [25]. The P 50 wave of P-ERG was suggested to re ect the function of retinal ganglion cells [26] while the N 95 wave was believed to be a speci c indicator of retinal ganglion cell function [27][28] and N 95 amplitude reduction implied function defects of retinal ganglion cells. There were many studies on patients with amblyopia in P-ERG changes, but results still remained inconsistent. Tepping et al. [21], Guttob et al. [15] and Hess et al. [16] found no signi cant difference between amblyopic eyes and the normal control eyes in either amplitude or latency of P 50 and N 95 . Tugcu B et al. [13] and Arden et al. [14], using the same stimulation patterns, found that among patients with amblyopia, there were no signi cant differences in the amblyopic eyes' P-ERG latency compared to the fellow eyes, but P 50 amplitude in the amblyopic eyes declined signi cantly when compared to the normal control eyes, indicating the decline of retinal ganglion cell function. Manny et al. [22], also under the same stimulation patterns, found that P 50 amplitude decreased in amblyopic eyes compared to the contralateral healthy eyes and pointed out that this may be due to the amblyopic eyes' retinal ganglion cell function decline. In this study, we used a low temporal frequency -high spatial frequency mode (HSLT) and a high temporal frequency -low spatial frequency mode (ISHT) to respectively bias parvocellular pathway and magnocellular pathway. These two visual transmission pathways are relatively separated from the retina to LGN, anatomically and functionally [23,24]. We found that both P 50 and N 95 wave amplitudes declined in patients with anisometropic amblyopia under the parvocellular pathway biased stimulation, while no magnocellular pathway impairments could be observed in anisometropic amblyopia. These results suggested that in the patients with anisometropic amblyopia, the damage might exist in some of retinal ganglion cells that were responsible for signal transmission of the parvocellular pathway.
Ganglion cell development requires the stimulus of a clear optical image [26]. In anisometropic amblyopia, the eyes' refractive state does not allow a clear image to be projected onto the retina. This may affect the normal development of the ganglion cells leading to structural and functional abnormalities, which may in turn affect the photoelectric signal conversion or signal transmission of the retina, resulting in amblyopia development. In strabismus amblyopia patients, neither GCC nor RNFL thickness was signi cantly different in the amblyopic eyes compared with the fellow eyes, and also no statistically signi cant difference existed in amplitude and latency of P 50 and N 95 when compared with the fellow eyes. Our results are consistent with the results of Altintas et al. [7] and Kee et al. [17]. These ndings also support the hypothesis that the mechanism of anisometropic amblyopia is different from strabismic amblyopia. The visual pathway damage of strabismic amblyopia may be mainly in the visual cortex [29], and the retina may be as normal as the fellow normal eyes.
Since the present study enrolled subjects who were all over the age of 18, the confounding factor of age has been excluded in comparison with previous studies. Axial length was not corrected during analysis because the authors did not nd a signi cant difference between the amblyopic eyes and the fellow eyes in AL. No signi cant correlation between axial length and OCT, P-ERG indicators was established. These results are consistent with the ndings of Szigeti A [11].

Conclusion
In this study, we found that in the amblyopic eyes in patients with anisometropic amblyopia, relative to the fellow healthy eyes, RNFL in the temporal sector was signi cantly thinner where the maculopapillary bundle is located, whereas RNFL thickness in the nasal, superior and inferior sectors was increased and the mean GCC layer was thickened as well. Functionally, P-ERG showed that the anisometropic amblyopia eyes had a reduction in the amplitude of P 50 and N 95 under the parvocellular pathway stimulated mode, indicating changes in the function of the retina in patients with anisometropic amblyopia was selective to parvocellular pathway. No statistically signi cant difference could be seen in OCT and ERG parameters among patients with strabismic amblyopia. These ndings also supported the hypothesis that the pathological mechanisms were different between anisometropic and strabismic amblyopia. The visual pathway damage of strabismic amblyopia may be mainly in the visual cortex.
In this study, we have some limitations. As the participants in this research is relatively low, and because of the functional limitations of PERG, its hard to distinct the parvocellular pathway and the magnocellular pathway very exactly. So, in the future of our work,we may nd some new way to distinct the two different pathways and include more cases.

Declarations
Ethics approval and consent to participate The study was approved by the ethics committee of the Anhui Medical University and followed the Declaration of Helsinki. Written informed consent was obtained from all subjects.

Consent to publish
Not applicable.

Availability of data and meterials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
We declare that we have no nancial or personal relationships with other people or organizations that could inappropriately in uence this work.